J. appl. Chem. Biotechnol.

1977, 27, 155-164

Method for the Determination of Oxygen Transfer Coefficients

(KLa)with the Correction for the Actual Cultivation Conditions
Jan PBca and Vratislav GrCgr
Department of Fermentation Chemistry and Technology, Institute of Chemical Technology,
166 28 Prague 6, Czechoslovakia
(Paper received8 July 1976 and accepted 22 October 1976)

A method for determination of the volumetric oxygen transfer coefficient KLa from
dynamic measurement data under conditions of both excess oxygen and oxygen-
limited growth of microorganisms is described. The KLa determination follows
from the integrated form of the oxygen balance equation and involves a correction
for the actual cultivation conditions during measurement.

1. Introduction
In studies of oxygen transfer from the gas phase into the growth medium during aerobic cultivation
it is necessary to know the value of the saturation concentration of dissolved oxygen and to observe
the dissolved oxygen concentration changes that can be considered as a measure of cell growth and
product formation. These parameters are of great importance in cases when oxygen limitation
can be achieved.
In the last decade considerable attention has been devoted to the determination of the volumetric
oxygen transfer coefficient. Many of these studies involve methods that can be applied only with
difficulty in a biological system (measurements are made under artificial conditions). Among these
belong the sulphite method,l-7 indirect biological methods, and static method.1°-15
For an estimation of KLa during the cultivation process the dynamic method and method of
steady-state oxygen balance can be used. Both these methods were compared by Tuffile and Pinhole
who concluded that the latter is better for viscous non-Newtonian broths. The method requires
simultaneous determination of oxygen in the outflowing gas and in the liquid phase. When the
total oxygen uptake rate by the culture is low this method is inaccurate.17
The dynamic response technique is often used in cell cultures because it enables estimation not
only of KLUbut also of respiration rate and the actual value of dissolved oxygen saturation concen-
tration. Studies on the dynamic method can be divided into two groups. In the first group
authorsls-24 studied this method from the chemical engineering standpoint without considering
the specificity of biological processes. The second group of authors developed methods that are
suitable to be applied for direct measurement during the cultivation of r n i c r o o r g a n i s r n ~ . ~ ~ - ~ ~
This study describes a method using the integrated form of the oxygen balance equation and
permitting the determination of the coefficient KLa and cell respiration rate with respect to the
changes in oxygen solubility that occur during cell cultivation.

2. Theory
2.1. Introduction
The general oxygen balance during continuous cell cultivation in an aerated and agitated fermenter
is given by equation (1).
dCL dP
+_ ~

dt
+
= KLU(C* - CL) D (CLR- CL)- q 0 2 ’ - k 5 (1)

155
156 J. P k a and V. Gr&

The first term on the right-hand side of equation (1) corresponds to the oxygen supply from the
gas phase and is influenced by agitator speed, gas flow rate and physico-chemical properties of the
cultivation broth. The next term, D(CLR- CL),expresses the oxygen supply from dissolved oxygen
present in the feed and dissolved oxygen removed in the liquid overflow. The other terms on the
right-hand side express the oxygen demand per unit volume of culture and oxygen consumption
for the oxidation of products, respectively.
To solve equation (1) the following assumptions were introduced:
1. Oxygen supply in the feed can be neglected.
2. qo,'=constant and KLa=constant in steady state of continuous culture and during a short
period of measurement in a batch cultivation.
3. Oxygen consumption for the oxidation of products is included in the cell oxygen demand
because it is impossible to estimate all metabolic products.
With these assumptions equation (1) can be rewritten in the form
+ -dCL
--=K~*I(C*-CL)-~O~-DCL
dt
Equation (2) was used for the determination of KLa.

2.2. K L determination
~ from the integrated form of the oxygen balance equation
The integrated form of the oxygen balance equation has already been used in a static method by
several a ~ t h 0 r s . l2~9 . For the evaluation of results obtained from a dynamic method, the integral
method was also used.27
The integral method is derived from equation (2). From the slope between points [tl, CLI] and
] value of qo2' can be determined (Figure 1)
[tz, C L ~the

Figure 1. The course of changes in dissolved

oxygen concentration with boundary points used for
90,' and KID determination.
fo 4 '2 '3 t
Time. t (5)

The driving force during oxygen absorption is a concentration difference which decreases with time.
During this process, represented by the portion [tg, C L ~to] [t4, C L ~in] Figure 1, the sign at the
left-hand side of equation (2) is positive. To integrate equation (2) it is necessary to assume that
during the operation of an interruption and following the start of reaeration C* = constant, go2'=
constant and KLa=constant. At steady state of continuous culture also D=constant. Then from
equation (2) it follows:
Determination of oxygen transfer coefficients 157

(4)

After integration of equation (4) we obtain

[(C*-qo,’lKLa)l(l+ D/KLa)- C L ~-I
In -ftl-t3
KLa+ D [(C*-qo,’lKLa)lil+ D/KLa)- C L ~ ]
At steady state in continuous culture, when

the term

corresponds to the actual value of dissolved oxygen at the testing time. This term expresses the
equilibrium condition for the oxygen supply, removal and demand in the fermenter.
The termqo,’/KLa is a function of the biomass concentration, physiological state of cells (expressed
by respiration rate), impeller speed and aeration rate. It can therefore be considered as a parameter
indicating the influence of the oxygen supply on the physiological state of the culture.
Substituting equation (6) in equation ( 5 ) and rearranging, equation (7) is obtained.

(7)

Equation (6) can be used as follows for the estimation of the actual value of dissolved oxygen
saturation concentration under the conditions of measurement. Substituting the values of qo,‘
obtained from equation (3) and KLa determined from equation (7) with the initial value of C* into
equation (6) the corrected value of c, (c)’
i.e. is obtained.
If (c)’=c, then the initial saturation concentration C* corresponds to the actual value of C,*
at the time of measurement. This equality could be theoretically achieved in a cell culture when no
metabolic products are secreted into the medium, nutrients in the growth medium are very slightly
depleted (e.g. at a very high dilution rate) and the carbon source is converted only to biomass,
C02 and HzO (e.g. by strictly aerobic microorganisms or at the beginning of a batch culture).
In actual cultures (C)’# C because of the depletion of nutrients and product formation. For this
reason the value of (C)’obtained by substituting C*, qo,’. D and KLa into equation (S),

is higher than that of the measured c.From this it follows that the actual value of C,* is smaller
than C* by the amount described by
AC= (C)’-c. (9)
The actual value of saturation concentration of dissolved oxygen can be obtained from the formula
C,* = C* - AC. (10)
Using Ca* from equation (10) the actual value of dissolved oxygen concentration can be determined.
This corrected value is substituted back into equation (7) and thereby the corrected coefficient
(Km)’for the actual experimental conditions is determined.
When the term DCL is equal to zero (batch operation) or is small compared with KLa(C*- CL)
and 40,’ in continuous culture equation (2) reduces to the form

12
158 J. Pica and V. Cr&r

Equation (6) simplifies to

SimiIarly equation (8) and the term D in equation (7) can be neglected. The further procedure for
C,* estimation remains the same.

2.3. Modified procedure for K L determination

~ under oxygen-limited conditions
Under conditions of oxygen limitation the concentration of dissolved oxygen in a cultivation medium
is very low or equal to zero. In this case, therefore, the usual dynamic method cannot be used.
To permit measurement under these conditions, the dynamic method was modified. This procedure
follows from Figure 2.

’I ’2
Time, t ( s )

-
?-.
.

-F
J
0

Figure 2. The course of changes in dissolved oxygen

concentration in the modified dynamic method.
(a) at time t > f without
~ aeration; (b) at time t > f 3
with initial values of n, Vg.
‘0 I‘ t2
Time, f (5)

First the oxygen demand of the cells is to be determined (Figure 2a). To attain a value of CL> 0
the agitator speed and aeration rate have to be increased. At the time tl the aeration is stopped with
simultaneous decrease of agitator speed to the original value (as at t < to). From the slope obtained
the total oxygen uptake rate qo2’ is determined by linear regression. The agitator speed and aeration
rate are again increased as before, and at time t 3 are reset to the initial values. The course of the
oxygen transfer driving force then has a decreasing character as follows from the descending part
of Figure 2b. Under these conditions equation (7) changes to the form
1 CI.3-c
Kw=---h---
t4-t3 cI.4-c
Determination of oxygen transfer coefficients 159

Simultaneously with equation (13), according to the procedure derived in the previous section 2.1,
equation (12) applies to the estimation of Ca*,too. Under conditions of oxygen limitation equation
(12) can have the following forms

To distinguish which of the conditions defined by equations (14) and (15) takes place is not easy
and depends on the nature of the measuring electrode. As was found using redox-potential measure-
ment,30 the oxygen pressure in a culture solution of aerobic bacteria is about 10-8 atm and in a
culture solution of facultative anaerobes about 10-lz atm. On the other hand, using oxygen probes
Harrison3I and Linton et aE.32 found a zero value of dissolved oxygen concentration, although
aeration was still proceeding. For the conditions described by equation (14) the value of KLa could
be determined from equation (13). With respect to the difficulties for Cdetermination as mentioned
above, equation (16) has to be used for KLUestimation, although this equation is strictly true only
for the conditions described by equation (15).
1 CL3
&a=-- In -
t4-t3 cL4

Comparing results obtained from equation (16) with those of equation (13) it was found that
since C L ~ S C
and C L ~ % >the, actual equilibrium concentration c can be neglected. The error
arising from neglecting C is smaller than 0.5 %.
When CLzO,it is necessary to determine the actual saturation concentration of dissolved oxygen
from equation (15), which gives C,* = (c)'.Comparing the corrected value &a)' with K L under ~
oxygen-limited conditions it is found that the difference is very small and therefore no correction
is necessary.

3. Experimental
3.1. Microorganism
The experimental microorganism was Klebsiellu uerogenes CCM 23 18 from the Czechoslovak Collec-
tion of Microorganisms.

3.2. Cultivation media

For the experimental cultures the modified synthetic medium according to Holme33 was used :
15.1 g Na2HP04.12Hz0, 3 g KHzP04, 5 g NHKI, 0.22 g Na~S04,1 ml20% MgS04.7H~0and
0.5 ml salt solution per litre. The salt solution contained: 0.18 g CoC12.6Hz0, 1.04 g CaC12.
6Hz0, 16.70 g FeC13.6Hz0, 0.18 g ZnS04.7Hz0, 0.16 g CuS04.5HzO and 0.15 g MnS04.4Hz0
per litre. In all experiments glucose at a concentration of 4 g/litre served as the carbon and energy
source. The inoculation medium contained a double amount of phosphates to ensure sufficient
buffering capacity. Individual components of inoculation and cultivation medium were sterilised
separately (including the solution of glucose) and mixed just before use.
3.3. Apparatus
The fermenter consists of a Simax glass cylinder; plates and inner equipment are made from stain-
less steel. Inner diameter is 106 mm, the ratio of working volume height to the diameter being 1.9.
Agitation is ensured by two six-blade open disk turbines, 50 mm in diameter, the impeller spacing
being 96 mm. The fermenter is fitted with four 10 mm wide baffles. Aeration intensity is adjusted
by two pressure-reducing valves maintaining a constant air flow rate at variable intake air pressure.
The air flow is gauged by a rotameter followed by manometer and thermometer. This arrangement
160 J. Pica and V. Grkgr

ensures a reproducible definition of aeration intensity. The air is forced into the fermenter by means
of a sparger, 1.6 mm in diameter, placed centrally below the lower impeller. Impellers are driven by
a direct current motor, their speed being controlled by a thyristor controller. The pH was kept
constant at 7.0 during cultivation by means of a pH-controller, 25 % NaOH being dispensed by a
solenoid valve in conjunction with a combined glass-silver chloride electrode. Temperature was
controlled through a relay system regulating cooling and heating units. The temperature was both
measured and maintained at 30_+0.3"C.The apparatus was sterilised in situ at 120°C for 2 h.
Dissolved oxygen and pH probes were sterilised by an ethanol solution.

3.4. Biomass determination

Biomass dry weight was determined by measuring the optical density on a spectral colorimeter
(Spekol, Zeiss, Jena, GDR) at 445 mm. In addition, a control gravimetric determination was
carried out. The samples were centrifuged, washed twice, and dried for 1 h at 70°C and for 2 h at
105°C.
3.5. Determination of the saturation concentration of dissolved oxygen
The concentration of dissolved oxygen was measured by an Oxytest MU-66 analyser (Develop-
mental Workshops, Czechoslovak Academy of Science) with an Ag-Ag/AgCl electrode covered
by a 12 pm thick polypropylene membrane. To ensure satisfactory regulation of oxygen supply dur-
ing the experiments, the calibration of the probe had to include a determination of the dynamic
effect of the liquid on the measurements. This determination was carried out both in water-air
and nutrient medium-air systems. Agitation at a speed of 200-1000 min-1 caused a 6 % deviation
in the saturating concentration measured. At aeration rates of 0.01-1.5 VVM under constant
agitator speed the deviation was 2%. These deviations are no longer negligible, especially when
measuring at low oxygen supply, and the probe had consequently to be calibrated in each experi-
ment for the actual agitator speed and aeration rate. This calibration eliminated the effect of
dynamic forces in the liquid flow due to agitation on the diffusion of oxygen through the probe
membrane.
The saturation concentration of dissolved oxygen was determined from the total pressure in the
fermenter above the liquid surface by means of the following formulae:

On measuring the decrease in oxygen solubility due to dissolved salts and sugar in the nutrient
medium it was found that the saturation concentration of dissolved oxygen in the medium is lower
by 2% than that in distilled water.
During the dynamic measurements that were made for Km determination the time courses of
dissolved oxygen changes were registered by a recorder TZ 21 (Laboratory Instruments, Prague)
connected to the oxygen analyser MU-66.

4. Results and discussion

Experiments were performed at two dilution rates, 0.96 h-I and 0.178 h-1, and in a range of oxygen
supply rates to verify the method described for the determination of KLa, actual saturation concen-
tration of dissolved oxygen and respiration rate of cells.
Figure 3 shows the time courses of dissolved oxygen concentration in the cultivation broth at the
Same impeller speed and aeration rate but at rather different dilution rates.
From equation (3), the values of qo2were determined. Under intense agitation/aeration conditions
(800 min-1, 1.O VVM) the term (- DCL)could be neglected and the results were determined from
equation (11). The significant difference in K m values obtained under conditions of the same
Determination of oxygen transfer coefficients 161

Figure 3. Actual courses of changes in dissohed

oxygen concentration at n=800 min-l, V,=
1 VVMi- , D = O . 96 h-1; ---, D = 0.178 h-l. I I
0 too 200
Time, 1 (5)

agitator speed and aeration rate but at rather different dilution rates results from changes in the
physiological state of cells (qo2)and physicochemical properties of the cultivation broth.34 The
procedures derived in the theoretical part can only be used when the bulk medium in the fermenter
is ideally mixed. In all cases of KLa measurements this condition was achieved. T o verify the derived
procedure of KLU estimation, a comparison with results obtained by the procedure of Bandyo-
padhyay et al.36was made and the two results, including the magnitude of differences, are sum-
marized in Table 1. It is evident that both methods give similar results. The maximum difference
(AKLa) obtained by both methods is 24%.
The comparison of saturation concentrations of dissolved oxygen determined by the procedure
derived with those presented by Bandyopadhyay et al.36 is given in Table 2. The results show a good
agreement. The maximum difference (AC,*) is f 2%.

Table 1. Comparison of KLa values determined from the integrated form

and by the method of Bandyopadhyay e l uLZ6at steady state of chemostat
culture

600 0.1 0.96 0.73 86.1 86.7 -0.70

600 0.3 0.96 0.83 97.6 94.3 +3.40
800 1.0 0.96 1.07 366.9 394.7 -3.16
800 1.0 0.178 2.80 164.6 158.2 +3.88

Table 2. Comparison of the actual saturation concentration of dissolved oxygen

obtained from the integrated form and by the method of Bandyopadhyay er d . Z 6
at steady state of chemostat cultures

D X c C* C,* C,B* ACa*

(h-1) (g 1-1) (mg 1-1) (mg I-l) (mg 1-l) (mg I-l) (%)

0.96 0.73 2.59 7.45 5.23 5.13 +1.91

0.96 0.83 2.58 7.35 6.04 6.11 -1.15
0.96 1.07 3.56 7.46 5.28 5.25 -0.56
0.178 2.80 3.31 7.38 5.13 5.21 -1.50
162 J. Pica and V. Grkgr

The value of KLa is influenced by the choice of the lower and upper points substituted in equations
(7) and (16) and the magnitude of and time of exposure to a dissolved oxygen concentration change
which can cause changes in the cell physiology.
The influence of the choice of boundary points was tested in all cases of KLa determination.
From the results it follows that the value of KLa is more sensitive to the value of the upper limit
(3-5%) than to the lower one (1.5-3%). To obtain correct values of KLa using this method it is
recommended to choose the lower limit value in the range of C~3=(0-0.15) e a n d the upper limit
value in the range of C L ~ (0.97-0.99)
= c. Under these conditions the error in the KLa value does
not exceed (0.02-0.08) K u . The precision of the determination depends on the absolute values of
KLa and the cell respiration rate. The higher the value of K w the lower must be the value of the
lower limit point and in the case of the upper limit vice versa. As shown by the results obtained,
these conclusions are valid for the evaluation by the dynamic method under excess oxygen and
oxygen limited conditions, respectively.
Applying the integrated form of the oxygen balance equation, the changes in the value of KLa,
which appear during the dynamic measurement, cannot be ascertained because this procedure
smooths the experimental data. Therefore this method gives an average value of KLa. To ascertain
whether KLa is a function of the concentration difference (C*- CL) during a specific test has a
meaning only from a theoretical standpoint of mass transfer.
The actual value of K L in~ a cultivation process can be obtained either by the dynamic method
or by the method of steady-state oxygen balance. N o method exists giving absolutely correct
results. The magnitude of the error cannot be exactly estimated because it is not known to what
extent the difference of K u obtained under the same hydrodynamic conditions (n, ri8> in distilled
water and in a cultivation medium is influenced by the physicochemical properties of a liquid
medium, ion selectivity of the covering membranes that affects the response of the oxygen electrode
and by the sharpness of the step-change in aeration rate.21 It can be seen that the average value of
KLa determined by the procedure described here is quite accurate for measurement during cultiva-
tion of microorganisms.
Several studiesls-20.23 have been devoted to elucidating the effects of the speed of oxygen elec-
trode response on the accuracy of the KLa determination. These experiments however were made
in water-air or growth medium-air systems only. Under these conditions the authors did not have
to solve problems arising in the cultivation of microorganisms. The method derived in this study is
independent of the speed of electrode response. On the other hand, the correction of the actual
value of the saturation concentration of dissolved oxygen under measurement conditions was
made. It is questionable which of the correction parameters mentioned is more significant under
real cultivation conditions. Therefore, the elucidation of some factors influencing the determination
of the absolute value of K u during cultivation of microorganisms is given below.
Difference of electrode response speeds that were found in air-water and air-growth medium
systems could be caused by: (1) purity of KCI electrolyte, (2) thickness of electrolyte layer between
the cathode and covering membrane (depending on electrode preparation-state of the membrane
stress), (3) quality of membrane material (impurity, embrittlemeat with age, ductility of material).
These factors influence the response speed in that part of the transport through the membrane
proper and the electrolyte layer to the cathode.
The oxygen transfer rate from a liquid phase to the membrane is influenced by the physico-
chemical properties of the cultivation broth and by dynamic forces of the liquid flow in the fermenter.
Among these physico-chemical properties belong : interfacial tension, viscosity, density and ionic
strength which is of particular significance in Newtonian systems of water-like viscosity.35 These
properties influence the changes in gas holdup, mean diameter of bubbles and coalescence and
thereby the values of K L too.~ The changes of physicochemical properties of the cultivation broth
in different steady states of the chemostat and during batch cultivation result from additions of
antifoam agents and changes in nutrient, metabolic products and cell concentrations. In addition, it
is necessary to consider the fouling of the probe membrane surface during long-term cultivations,
too.
The correct determination of the absolute value of K w requires electrode calibration immediately
Determination of oxygen transfer coefficients 163

before and after dynamic measurements. In addition, this calibration has to be performed under
the same hydrodynamic conditions as the measurement proper, i.e. in a geometrically identical
vessel and at the same impeller speed and aeration rate. Under cultivation conditions this require-
ment is difficult to achieve. It would be necessary to determine first the probe response speed in a
fermenter filled with a growth medium, then take out the probe (if it is non-sterilisable), sterilise
the medium in a fermenter, cool, start the cultivation and make a measurement during the process.
In the case of a thermal probe sterilisation changes in the probe response speed may also occur.
For KLU determination at steady state in continuous cultivation, the probe has to be placed in
the fermenter from the start of the batch process to avoid the medium becoming contaminated.
It means on the one hand that there is a long time interval from the calibration of the probe to the
time of measurement and, on the other hand, that the probe is exposed for a long time to the culti-
vation broth. As no membrane is strictly semipermeable, some other ions than oxygen can penetrate
into the electrolyte. Therefore it is difficult to avoid changes in response speed caused by the deple-
tion of the electrolyte and cathode poisoning. To prevent these last factors it would be better to use
a thicker membrane. On the other hand however, this results in undesirable delay of probe response.
In respect to the above mentioned circumstances it is questionable which factor is more important
for KLa determination under cultivation conditions: the probe response speed or the changes in
the saturation concentration of dissolved oxygen.
The procedure of KLa estimation described in this study compared with that of Bandyopadhyay26
has the advantage that dCr,/dt need not be calculated. Unlike the method presented by Fujio et 4 2 7
this procedure involves a modified numerical iteration that permits correction to the actual satura-
tion concentration of dissolved oxygen during the determination of KLU in cultures of micro-
organisms. In addition, the procedure for KLUdetermination under conditions of oxygen limitation
was derived, examined and verified. Both procedures give sufficiently accurate results in the range
of KLa studied. From the comparison of results obtained, which was made on the basis of corres-
ponding relations between X , go2, c
and KLU and the values presented by Topiwala and
Sinclair,2,36 good agreement can be seen. From this it follows that the derived methods for KLU
determination are suitable for practical applications. Similarly the values of Ca* estimated are in
good agreement with results published by Phillips and Johnson.37

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Symbols
c* Saturation concentration of dissolved oxygen in growth medium before inoculation
(mg 1-9
C*8 Actual saturation concentration of dissolved oxygen in cultivation broth at the time
of measurement (mg 1-1)
Value of C*acalculated by method of ref. (26)
Difference between Ca* and c8B* (%)
Dissolved oxygen concentration in cultivation broth (mg 1-1)
Dissolved oxygen concentration in input medium (mg 1-l)
Measured concentration of dissolved oxygen at the start of the dynamic test (mg 1-1)
Actual concentration of dissolved oxygen at the start of the dynamic test (mg 1-1)
Difference defined by equation (9)
Dilution rate (h-1)
Henry's constant (Pa)
Pressure above the liquid level in the fermenter (Pa)
Rate constant in equation (1)
Volumetric oxygen transfer coefficient (h-1)
Value of K m calculated by method of ref. (26)
Difference between KLa and KLaB (%)
Molecular weights of oxygen and water, resp.
Agitator speed (min-1)
Concentration of metabolic products (g 1-1)
Partial pressure of oxygen in gas phase (Pa)
Barometric pressure (Pa)
Respiration rate of cells (mmol g d.w.-' h-l)
Total oxygen uptake rate (mmol l-1 h-1)
time (s)
Volume of gas/volume of liquid/min (min-1)
Volumetric gas flow rate to the fermenter (VVM) (min-l)
Cell dry weight (g 1-1)
Water density in a manometric tube at the temperature of measurement and under
normal conditions, resp. (kg m-3)
51 Water density in fermenter at cultivation temperature (kg m-3)